Figure 1.

Characterization of Fab1 point mutants. (A) Schematic representation of the Fab1 domain structure depicting the FYVE domain, the CCT, the CCR domains that together form the GroL region, and the lipid kinase domain. Mutations or truncations were introduced into each domain as indicated. (B) Amino acid sequence alignment of a conserved area in the CCT and CCR domains of several Fab1 orthologues: mm, Mus musculus; Hs, Homo sapiens; Sc, S. cerevisiae; Af, Aspergillus fumigatus; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans. The asterisk (*) denotes conserved residues, whereas the colon (:) and the period (.) indicate near-identical or similar residues, respectively. Enclosed residues were mutated as indicated above. (C) Cellular levels of PtdIns(3,5)P₂ as a percentage of total PtdIns in cells expressing the indicated Fab1 mutants. (D) Temperature-sensitive growth of fab1Δ cells expressing Fab1 mutants. Serial dilutions of cultures grown at 26 or 38°C for three days. (E) Vacuolar morphology of fab1Δ cells expressing the designated fab1 mutants. Cells were labeled with FM4-64 to demarcate the vacuolar limiting membrane. The FM4-64 and DIC images were merged as a grayscale palette. Bar, 5 μm.